Density microfluidic equipment and method for separating and purifying high-concentration leukocytes, platelets and cytokines

Abstract

The invention provides density microfluidic equipment and a method for separating and purifying high-concentration leukocytes, platelets and cytokines, and relates to the technical field of density microfluidics. Comprising a wide shunting pipeline, m inlets are formed in the front end of the wide shunting pipeline, n outlets are formed in the rear end of the wide shunting pipeline, m is larger than or equal to 3, and n is smaller than m by 1; the inlet and the outlet are sequentially marked from top to bottom, and the height of the outlet with the same serial number is not higher than the height of the inlet with the corresponding serial number and not lower than the height of the inlet with the next serial number. The microfluidic equipment can continuously sort cells according to the density of each component in blood to obtain high-concentration autologous white blood cells, blood platelets and cell factor solutions and keep the activity of the high-concentration autologous white blood cells, blood platelets and cell factor solutions; compared with a traditional centrifugal method and an existing micro-fluidic method, a simpler, milder and continuous high-flux and high-activity cell sorting and solution preparation method can be realized, so that a solution which is higher in concentration, stronger in functionality and rich in high-activity white blood cells, blood platelets and cell factors is generated.

Description

Technical Field

[0001] The present invention relates to the field of density microfluidics technology, in particular to a density microfluidics device and a method for separating and purifying high-concentration leukocytes, platelets and cytokines. Background Art

[0002] In medical treatment, autologous leukocytes, platelets and other cytokines can be used to treat aseptic inflammatory diseases such as arthritis and tendonitis. It is a relatively new cell therapy with broad application prospects. Compared with the widely used platelet-rich plasma therapy on the market, this solution contains concentrated leukocytes and cytokines. Cytokines provide short-acting anti-inflammatory and analgesic effects, and leukocytes provide long-acting anti-inflammatory factor release, thereby providing long-term inflammation and pain relief, as well as regeneration of injured tissues such as soft tissue or bone tissue.

[0003] In existing technologies, there is no specific separation device for solutions of autologous white blood cells, platelets, and cytokines, making extraction very difficult. While platelets can be obtained clinically by centrifuging platelet-rich plasma, there is no clinical method for extracting an "autologous white blood cell and cytokine solution."

[0004] The primary blood separation methods currently available on the market are density gradient separation methods based on centrifuges and separation gels. These methods have their limitations: 1. Prolonged high-speed centrifugation is detrimental to white blood cell viability; 2. Since the collection of the white blood cell layer inevitably removes the separation gel layer, its toxic side effects in humans when used as a drug are unknown, and a washing step is required to remove the medium; 3. Lymphocyte separation media Ficoll and Histopaque-1077 are the most commonly used methods for separating the white blood cell layer, but their density is insufficient, and they can only separate a portion of white blood cells (mononuclear leukocytes, such as lymphocytes). Separating the entire white blood cell layer and maintaining a stable ratio of various white blood cell components (mononuclear leukocytes and polynuclear leukocytes) are crucial for maintaining the microenvironment in which white blood cells survive, which directly determines the activity of the solution after autologous solution reinfusion. Ficoll and Histopaque-1077 are products of Sigma, and polysucrose and sodium diatrizoate solution have a density of 1.077 g / ml; 4. Sodium diatrizoate solution, as an iodine-containing benzoate contrast agent, cannot be used for therapeutic purposes due to its radioactivity.

[0005] Microfluidics is a new type of particle separation method that does not require a centrifuge. In the microfluidic system, blood is kept in a laminar flow state, reducing physical damage to sensitive biological molecules such as cells or proteins. , such as spiral channel inertial separation technology. However, since current microfluidics technology is mainly based on medical research or detection purposes, and can only separate a single type of cell, such as white blood cells, from a small amount of blood, it cannot be used as a therapeutic platform for the following reasons: Since the amount of blood required for cell therapy is much greater than that required for diagnosis, there is an urgent need for ultra-high throughput methods. The current cell diagnostic platform has limited flow, such as the fastest commercial inertial microfluidic chip of 500μL / min [Lu, Xiaoguang, Mahnoush Tayebi, and YeAi. "A low-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood." Electrophoresis 42.21-22(2021):2281-2292]. A single microchannel is not enough for commercially relevant flow. When the cell concentration is higher than 10 7 When the number of cells / mL is 1000, the efficiency of the system drops significantly because the number of red blood cells far exceeds the number of white blood cells typically useful in treatment, with a ratio of approximately 1000:1. When whole blood enters the microchannel, the large number of red blood cells tends to stack, squeeze, and fill the channel, causing blockage. This can also cause blockage in such a small hydraulic diameter channel. Moreover, highly diluted blood means longer processing time. Currently, to separate cells from whole blood, most blood pretreatment steps require a lysis step to reduce the volume of red blood cells. However, the lysis buffer is toxic, and non-toxic treatment methods are necessary. In addition, because the blood is heavily diluted in the chip, the active ingredients are also diluted and cannot be used in treatment. Summary of the Invention

[0006] The present invention provides a density microfluidic device and a method for separating and purifying high-concentration white blood cells, platelets and cytokines, which can separate and purify high-concentration white blood cells and cytokines in a simpler, gentler and continuous high-throughput and high-activity cell sorting and solution preparation method.

[0007] The specific technical solution is a density microfluidic device, including: a wide diversion channel, with m inlets at the front end and n outlets at the rear end, m≧3, and n is 1 less than m; the inlets and outlets are numbered from top to bottom, and the height of the outlet with the same number is not higher than the height of the inlet with the corresponding number, and not lower than the height of the inlet with the next number.

[0008] Furthermore, the wide diversion pipe is provided with 4 inlets at the front end and 3 outlets at the rear end. The inlets are referred to as the first front pipe port, the second front pipe port, the third front pipe port and the fourth front pipe port from top to bottom, and the outlets are referred to as the first rear pipe port, the second rear pipe port and the third rear pipe port from top to bottom. The height of the first rear pipe port is not higher than the height of the first front pipe port and not lower than the height of the second front pipe port, the height of the second rear pipe port is not higher than the height of the second front pipe port and not lower than the height of the third front pipe port, and the height of the third rear pipe port is not higher than the height of the third front pipe port and not lower than the height of the fourth front pipe port.

[0009] Furthermore, the wide shunt channel is longer than 22mm, no higher than 1mm, and has a width of 1mm-2000mm. A wide shunt channel with a length greater than 22mm allows red blood cells to reach the lower outlet, and a height no higher than 1mm allows the blood to maintain a laminar flow state in the microfluidic system, which can reduce physical damage to sensitive biological molecules such as cells or proteins. On this basis, the width of the channel is increased by 1mm-2000mm to improve the microfluidic flux. The flow rate is 5ul / min to 10ml / min. Due to the small height, the velocity gradient of the fluid in the vertical direction is small when the channel is wide and shallow. The small height limits the movement of the fluid perpendicular to the flow direction, which helps to reduce the generation of eddies and turbulence, so that the blood still maintains a laminar flow state.

[0010] Furthermore, all inlets and outlets are connected to pipes, each inlet is connected to a pipe with a precision injection pump and a peristaltic pump, each outlet is connected to a pipe with a spectrophotometer, and all precision injection pumps, peristaltic pumps and spectrophotometers are connected to a controller.

[0011] Furthermore, the pipeline connected to the second rear pipe port is provided with an enrichment filter membrane or is directly connected to an inertial focusing microfluidic channel.

[0012] Furthermore, the wide branch duct is made of laser-cut PMMA sheet.

[0013] Furthermore, wide diversion channels can also be made on silicon chips through deep etching technology. One method is to carve a convex channel on the silicon chip, then cast PDMS to form a concave channel, and finally bond the PDMS to the glass; another method is to directly bond the concave channel carved from PDMS to the silicon chip.

[0014] The steps for separating and purifying high-concentration white blood cells and cytokines using the above-mentioned microfluidic device are as follows:

[0015] S1. Blood is fed into the wide shunt channel from the top inlet, and separation fluids of different densities are injected into the wide shunt channel from different inlets.

[0016] S2. The separation fluid and blood flow into the wide diversion pipe at a certain speed at the same time. Different substances in the blood will stay in the separation fluid with corresponding density according to their own density.

[0017] S3, plasma flows out from the top outlet, white blood cells, platelets and cytokines flow out from the middle outlet, and red blood cells flow out from the bottom outlet.

[0018] S4. The separated white blood cells, platelets and cytokine solutions are first passed through a spectrophotometer. The flow rates of the blood and separation solution are adjusted according to the white blood cell concentration, and the density of the separation solution is fine-tuned.

[0019] S5. The leukocyte, platelet and cytokine solutions reaching the required concentration are then concentrated through an enrichment filter or an inertial focusing microfluidic channel.

[0020] Further, the density gradient solution was prepared: Percoll is a non-toxic aqueous solution of 15-30 nm colloidal silica coated with polyvinyl pyrrolidone, which has a relatively high density and is added to the buffer to adjust the density of the solution.

[0021] Furthermore, glucose is added to the Percoll buffer to prepare a 1%-10% dextran solution with a molecular weight of 100-1500. The dextran solution specifically induces aggregation of red blood cells in the blood, increases the concentration of red blood cells, and thus accelerates the sinking speed of red blood cells.

[0022] The microfluidic device of the present invention can continuously sort cells according to the density of each component in the blood without the use of a centrifuge or other important hardware; the microfluidic device of the present invention uses density microfluidic technology to separate blood components to obtain high-concentration autologous white blood cells, platelets and cytokine solutions while maintaining their activity; compared with traditional centrifugation and existing microfluidic methods, the present invention can achieve a simpler, gentler and continuous high-throughput high-activity cell sorting and solution preparation method, thereby producing a solution with higher concentration, stronger functionality, and rich in highly active white blood cells and cytokines for the treatment of diseases such as arthritis and tissue muscle damage repair; high flow is reflected in a flow rate greater than 10mL / min, and high concentration is higher than 10 7 cells / mL to meet the needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 This is a schematic diagram of the main pipelines of the microfluidic device of the present invention and its separation and purification of high-concentration white blood cells and cytokines.

[0025] Figure 2 is a schematic diagram of the inertial focusing microfluidic channel in the present invention,

[0026] 1. Wide diversion pipe, 2. First front pipe opening, 3. Second front pipe opening, 4. Third front pipe opening, 5. Fourth front pipe opening, 6. First rear pipe opening, 7. Second rear pipe opening, 8. Third rear pipe opening. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and the accompanying drawings. Here, the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0028] The following is a description of the present invention with reference to the accompanying drawings:

[0029] Combine Figure 1 It is understood that the density microfluidic device includes: a wide diversion channel 1, the wide diversion channel 1 is provided with m inlets at the front end and n outlets at the rear end, m≧3, n is 1 less than m; the inlets and outlets are numbered from top to bottom, and the height of the outlet with the same number is not higher than the height of the inlet with the corresponding number, and is not lower than the height of the inlet with the next number.

[0030] In one embodiment, the wide diversion pipe 1 is provided with four inlets at the front end and three outlets at the rear end. The inlets are referred to as the first front pipe port 22, the second front pipe port 33, the third front pipe port 44 and the fourth front pipe port 55 from top to bottom, and the outlets are referred to as the first rear pipe port 6, the second rear pipe port 7 and the third rear pipe port 8 from top to bottom. The height of the first rear pipe port 6 is not higher than the height of the first front pipe port 2 and not lower than the height of the second front pipe port 3. The height of the second rear pipe port 7 is not higher than the height of the second front pipe port 3 and not lower than the height of the third front pipe port 4. The height of the third rear pipe port 8 is not higher than the height of the third front pipe port 4 and not lower than the height of the fourth front pipe port 5.

[0031] In one embodiment, the wide shunt channel 1 is longer than 22 mm, not more than 1 mm in height, and has a width of 1 mm to 2000 mm. A wide shunt channel with a length greater than 22 mm allows red blood cells to reach the lower outlet, and a height of not more than 1 mm allows the blood to maintain a laminar flow state in the microfluidic system, which can reduce physical damage to sensitive biological molecules such as cells or proteins. On this basis, the width of the channel is increased by 1 mm to 2000 mm to improve the microfluidic flux. The flow rate is 5 ul / min to 10 ml / min. Due to the small height, the velocity gradient of the fluid in the vertical direction is small when the channel is wide and shallow. The small height limits the movement of the fluid perpendicular to the flow direction, which helps to reduce the generation of eddies and turbulence, so that the blood still maintains a laminar flow state.

[0032] In one embodiment, all inlets and outlets are connected to pipes, and a precision injection pump and a peristaltic pump are installed on the pipe connected to each inlet, and a spectrophotometer is installed on the pipe connected to each outlet. All precision injection pumps, peristaltic pumps and spectrophotometers are connected to a controller.

[0033] In one embodiment, the pipeline connected to the second rear pipe port 7 is provided with an enrichment filter membrane or is directly connected to an inertial focusing microfluidic channel.

[0034] Combine Figure 2 It is understood that the inertial focusing microfluidic channel is an asymmetric inertial focusing curved separation microchannel structure, also known as an asymmetric curved flow channel, which is used to enrich cell components in the concentration tube.

[0035] The asymmetric curved flow channel consists of multiple narrow turns and multiple wide turns. The width of the narrow turn is 350 μm, the width of the wide turn is 650 μm, and the average curvature radius of the narrow turn and the wide turn is 325 μm and 890 μm, respectively. This geometric shape causes the Dean resistance of the asymmetric system to be 8 times larger in small-radius turns than in large turns. The end of the asymmetric curved flow channel forms an outer outlet for the flow of buffer solution and an inner outlet for the circulation of cellular components.

[0036] Regarding the design description of the channel width and curvature radius at narrow turns and wide turns in asymmetric curved channels, there is a correlation, and the formula is as follows:

[0037]

[0038] Where r is the average curvature radius of the channel, r1 is the curvature radius of the narrow bend, r2 is the curvature radius of the wide bend, and D h is the hydraulic diameter of the channel, a c is the cutoff diameter of the particle, that is, the particle diameter that the channel can separate.

[0039] In the experiment, we obtained a set of feasible parameters: In this application we set a c The width of the narrow bend is 325μm, the height is 890μm, the width at the bend with a small curvature radius is 350μm, and the width at the bend with a large curvature radius is 650μm. The average curvature radii of narrow bends and wide bends are 325μm and 890μm respectively.

[0040] In one embodiment, the wide branch pipe 1 is made of laser-cut PMMA sheet.

[0041] In one embodiment, wide shunt channels can also be fabricated on silicon wafers using deep etching techniques. One method involves carving convex channels on the silicon wafer, then casting a PDMS mold to create concave channels, and finally bonding the PDMS to the glass. Another method involves directly bonding the PDMS-carved concave channels to the silicon wafer.

[0042] The steps for separating and purifying high-concentration white blood cells and cytokines using the above-mentioned microfluidic device are as follows:

[0043] S1, blood is fed into the wide diversion channel 1 from the top inlet, and separation fluids of different densities are fed into the wide diversion channel 1 from different inlets.

[0044] S2, the separation liquid and blood flow into the wide diversion pipe 1 at a certain speed at the same time. Different substances in the blood will stay in the separation liquid with corresponding density according to their own density.

[0045] S3, plasma flows out from the top outlet, white blood cells, platelets and cytokines flow out from the middle outlet, and red blood cells flow out from the bottom outlet.

[0046] S4. The separated white blood cells, platelets and cytokine solutions are first passed through a spectrophotometer. The flow rates of the blood and separation solution are adjusted according to the white blood cell concentration, and the density of the separation solution is fine-tuned to achieve more accurate separation.

[0047] S5. The leukocyte, platelet and cytokine solutions reaching the required concentration are then concentrated through an enrichment filter or an inertial focusing microfluidic channel.

[0048] In one embodiment, a density gradient solution is prepared as follows: Percoll is a non-toxic aqueous solution of 15-30 nm colloidal silica coated with polyvinyl pyrrolidone, which has a relatively high density and is added to a buffer to adjust the density of the solution without significantly affecting the ionic strength and osmotic pressure of the solution.

[0049] In one embodiment, to accelerate the sinking rate of red blood cells, glucose can be added to the Percoll buffer to prepare a 1%-10% dextran solution with a molecular weight of 100-1500. This is used to induce aggregation of red blood cells, resulting in a greater density difference between red blood cells and other components, thereby accelerating the sinking rate of red blood cells.

[0050] Example 1: A density microfluidic device, comprising: a wide shunt pipe 1, wherein the wide shunt pipe 1 is provided with m inlets at the front end and n outlets at the rear end, m≧3, and n is 1 less than m; the inlets and outlets are numbered from top to bottom, and the height of the outlet with the same serial number is not higher than the height of the inlet with the corresponding serial number, and is not lower than the height of the inlet with the next serial number; all inlets and outlets are connected to pipes respectively, and a precision injection pump and a peristaltic pump are installed on the pipe connected to each inlet to achieve stable and constant sample liquid flow control, and a spectrophotometer is installed on the pipe connected to each outlet, and all precision injection pumps, peristaltic pumps and spectrophotometers are connected to a controller.

[0051] Principle explanation: If a fluid contains a cell mixture, the cells will not only move horizontally through the channel with the fluid flow, but also move vertically to their isopycnic points due to buoyancy. Under laminar flow conditions, these fluids form a micrometer-scale density gradient flowing along the horizontal channel.

[0052] During application, multiple separation fluids of varying densities are injected into a wide diversion channel 1. These separation fluids flow together under laminar flow conditions to form a continuous micrometer-scale density gradient. In this miniaturized density gradient, cells need to travel a very short distance before reaching the isopycnic point, and under Earth's 1g gravitational acceleration, they travel this micrometer-scale distance in just a few seconds. When the channel splits, cells of varying densities gather at different outlets, ready for counting or further analysis. This technology can continuously sort a stream of cells based solely on their density, unaffected by other physical properties of the cells, and can separate cells of interest from other cells with higher throughput and milder conditions than existing tools.

[0053] Example 2: To separate leukocytes, platelets and cytokines. Based on Example 1, combined Figure 1 It is understood that the wide diversion pipe 1 is provided with 4 inlets at the front end and 3 outlets at the rear end. The inlets are referred to as the first front pipe port 2, the second front pipe port 3, the third front pipe port 4 and the fourth front pipe port 5 from top to bottom, and the outlets are referred to as the first rear pipe port 6, the second rear pipe port 7 and the third rear pipe port 8 from top to bottom. The height of the first rear pipe port 6 is not higher than the height of the first front pipe port 2 and not lower than the height of the second front pipe port 3, the height of the second rear pipe port 7 is not higher than the height of the second front pipe port 3 and not lower than the height of the third front pipe port 4, and the height of the third rear pipe port 8 is not higher than the height of the third front pipe port 4 and not lower than the height of the fourth front pipe port 5; the pipe connected to the second rear pipe port 7 is provided with an enrichment filter membrane or is directly connected to the inertial focusing microfluidic channel; the densities of the injected separation liquid are 1.015g / ml, 1.07g / ml, 1.085g / ml, and 1.11g / ml respectively.

[0054] When blood is input into the wide diversion channel 1 from the top inlet, separation fluids of different densities are injected into the wide diversion channel 1 from different inlets, with the separation fluid of low density being injected from the top inlet and the separation fluid of highest density being injected from the bottom inlet. The flow rate at the inlet of each fluid is 12 μL / min, and the combined flow rate is 48 μL / min. The separation fluid and blood flow into the wide diversion channel 1 at a certain speed at the same time. Different substances in the blood will stay in the separation fluid of corresponding density according to their own density, with red blood cells at the bottom layer and white blood cells and cytokines in the middle layer, thereby achieving cell separation. When blood cells are added to the liquid with the lowest density, that is, the top fluid contains a mixture of blood cells, the flowing white blood cells will quickly sink to the interface between the 1.070 and 1.085 g / mL fluids, where they are neutrally buoyant, and the average density of the flowing white blood cells is ρ = 1.080 g / mL. The flowing red blood cells sink to the interface between the 1.085 and 1.110 g / mL liquids, and the average density of the flowing red blood cells is ρ.

[0055] =1.110g / mL; plasma will remain at the uppermost interface. When the channel splits, plasma flows out from the uppermost outlet, white blood cells, platelets, and cytokines flow out from the middle outlet, and red blood cells flow out from the bottom outlet. The separated white blood cell, platelet, and cytokine solutions are first passed through a spectrophotometer. The flow rates of the blood and separation fluid are adjusted according to the white blood cell concentration, and the density of the separation fluid is fine-tuned to achieve more accurate separation. The white blood cell, platelet, and cytokine solutions that reach the required concentration are then concentrated through an enrichment filter or an inertial focusing microfluidic channel. Inertial focusing microfluidic channels such as Figure 2 shown.

[0056] The microfluidic device in the present invention uses density microfluidic technology to separate blood components to obtain high-concentration autologous white blood cells, platelets and cytokine solutions while maintaining their activity. Compared with traditional centrifugation and existing microfluidic methods, the present invention can achieve a simpler, gentler and continuous high-throughput and high-activity cell sorting and solution preparation method, thereby producing a solution with higher concentration, stronger functionality, and rich in highly active white blood cells, platelets and cytokines for the treatment of diseases such as arthritis and tissue and muscle damage repair.

[0057] Calculate the trajectories followed by cells of different sizes and densities as they flow through the chip:

[0058] 1. The first formula (horizontal velocity distribution):

[0059]

[0060] in:

[0061] ·u x : velocity distribution of the fluid in the x direction (horizontal direction);

[0062] ·u max : Maximum velocity of the fluid on the centerline of the pipeline;

[0063] y: vertical distance from the bottom of the pipe;

[0064] h: total height of the pipeline.

[0065] 2. The second formula (sedimentation velocity):

[0066]

[0067] in:

[0068] ·v y : the sedimentation velocity of the particle in the y direction (vertically downward);

[0069] ·ρ o : density of particles;

[0070] ·ρ f : density of the fluid;

[0071] g: acceleration due to gravity, usually 9.8 m / s 2 ;

[0072] r: radius of the particle;

[0073] μ f : Viscosity of the fluid.

[0074] Calculations show that an 8mm channel allows white blood cells to reach the middle outlet, while a 22mm channel allows red blood cells to reach the lower outlet. Experiments have shown that extending the channel can further improve separation efficiency, and that widening the channel laterally can increase flow and separation speed while maintaining laminar flow. The channel is designed to be 25mm long, 1mm high, and 10mm wide.

[0075] Preparation of density gradient solutions: Percoll is a non-toxic aqueous solution of 15-30 nm colloidal silica coated with polyvinylpyrrolidone. It has a relatively high density and is added to a buffer solution to adjust the density without significantly affecting the solution's ionic strength and osmotic pressure. To accelerate the sinking of red blood cells, glucose can be added to the Percoll buffer to create a 1%-10% dextran solution with a molecular weight of 100-1500.

[0076] Fabrication of the wide shunt channel 1: The wide shunt channel can also be fabricated on a silicon wafer using deep etching techniques. One method involves carving a convex channel on the silicon wafer, then casting a PDMS mold to create a concave channel, and finally bonding the PDMS to the glass. Another method involves directly bonding the carved concave PDMS channel to the silicon wafer. Alternatively, the wide shunt channel 1 can be fabricated by laser-cutting a PMMA sheet, then drilling holes to access the channel.

[0077] The medium-density microfluidic device in this application can efficiently separate cells with a separation rate of 90%. It has the characteristics of high flow and high concentration. High flow is reflected in the flow rate greater than 10mL / min, and high concentration is higher than 10 7 cells / mL to meet the needs.

[0078] The preparation process of the autologous leukocyte and cytokine solution is as follows: 20 ml of the patient's autologous blood is drawn; the blood and buffer are pumped into the medium-density microfluidic device of this application to obtain approximately 3 ml of a mixed solution of autologous leukocytes and cytokines; the mixed solution is passed through a concentration tube or 8KDa filter paper to obtain 1.5 ml of concentrated autologous leukocyte, platelet and cytokine solution; this solution can be directly injected into the joint cavity or ligament injury site for the treatment of chronic traumatic aseptic inflammatory diseases, such as arthritis and tendonitis.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Density microfluidic device, characterized in that, include: Wide diversion pipe, the front end of the wide diversion pipe is provided with m inlets and the rear end is provided with n outlets, m≧3, n is 1 less than m; the inlets and outlets are numbered from top to bottom, and the height of the outlet with the same number is not higher than the height of the inlet with the corresponding number, and is not lower than the height of the inlet with the next number.

2. The density microfluidic device according to claim 1, characterized in that The wide diversion pipe is provided with 4 inlets at the front end and 3 outlets at the rear end. The inlets are referred to as the first front pipe port, the second front pipe port, the third front pipe port and the fourth front pipe port from top to bottom, and the outlets are referred to as the first rear pipe port, the second rear pipe port and the third rear pipe port from top to bottom. The height of the first rear pipe port is not higher than the height of the first front pipe port and not lower than the height of the second front pipe port, the height of the second rear pipe port is not higher than the height of the second front pipe port and not lower than the height of the third front pipe port, and the height of the third rear pipe port is not higher than the height of the third front pipe port and not lower than the height of the fourth front pipe port.

3. The density microfluidic device according to claim 2, characterized in that: The length of the wide diversion pipe is greater than 22mm, the height is no more than 1mm, and the width is 1mm-2000mm.

4. The density microfluidic device according to any one of claims 1 to 3, characterized in that: All inlets and outlets are connected to pipes respectively. A precision injection pump and a peristaltic pump are installed on the pipe connected to each inlet respectively. A spectrophotometer is installed on the pipe connected to each outlet respectively. All precision injection pumps, peristaltic pumps and spectrophotometers are connected to the controller.

5. The density microfluidic device according to claim 4, characterized in that: The pipeline connected to the second rear pipe port is provided with an enrichment filter membrane or is directly connected to an inertial focusing microfluidic channel.

6. The density microfluidic device according to claim 4, characterized in that: The wide diverter duct is made from laser-cut PMMA sheet.

7. The density microfluidic device according to claim 4, characterized in that: The wide shunt channel is made on the silicon chip through deep etching technology. One method is to carve a convex channel on the silicon chip, then pour PDMS to mold a concave channel, and finally bond the PDMS to the glass. Another method is to directly bond the concave channel carved from PDMS to the silicon chip.

8. A method for separating and purifying high-concentration leukocytes, platelets and cytokines, characterized in that: Here are the steps: S1. Blood is fed into the wide shunt channel from the top inlet, and separation fluids of different densities are injected into the wide shunt channel from different inlets. S2. The separation fluid and blood flow into the wide diversion pipe at a certain speed at the same time. Different substances in the blood will stay in the separation fluid with corresponding density according to their own density. S3, plasma flows out from the top outlet, white blood cells, platelets and cytokines flow out from the middle outlet, and red blood cells flow out from the bottom outlet. S4. The separated leukocytes, platelets and cytokine solutions are first concentrated through an enrichment filter or an inertial focusing microfluidic channel. S5. The concentrated white blood cells, platelets, and cytokine solution is then passed through a spectrophotometer. The flow rates of the blood and separation solution are adjusted according to the white blood cell concentration, and the density of the separation solution is fine-tuned.

9. The method for separating and purifying high-concentration leukocytes, platelets and cytokines according to claim 8, characterized in that: Preparation of density gradient solution: Percoll is a non-toxic aqueous solution of 15-30 nm colloidal silica coated with polyvinyl pyrrolidone. It has a relatively high density and is added to the buffer to adjust the density of the solution.

10. The method for separating and purifying high-concentration leukocytes, platelets and cytokines according to claim 9, characterized in that: Glucose is added to the Percoll buffer to prepare a 1%-10% dextran solution with a molecular weight of 100-1500.

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